The chick embryo, in the initial stages of its development from fertilisation to cleavage, has not been amenable to experimental intervention on account of the bulk, fragility and relative inaccessibility of the ovum. This problem has been discussed in recent reviews on the possible routes of transfer of exogenous genes into birds (Freeman and Messer, 1985; Crittenden and Salter, 1986). Perry (1986a,b) has taken the alternative view and suggested that genetic manipulation of the avian ovum is practicable. The aim of devising a complete culture system for the chick embryo was to provide a means of rearing the manipulated ovum to maturity. A method has now been established for in vitro culture to an intermediate stage of embryonic development, and progress is being made on this account. The technique will have applications not only in the genetic engineering of poultry, but also in the investigation of fundamental mechanisms of avian development and in the study of deleterious traits. Moreover, it will afford a desirable alternative to surgical intervention in the laying hen.
The chick embryo originates in the germinal disc, a small region of cytoplasm situated at the animal pole of the ovum (the familiar yolk). During the first third of its development, the embryo remains floating at the surface of the yolk whilst the extra-embryonic membranes grow around the yolk and become vascularised. In the remaining period of development, the embryo grows at the expense of the food reserves in the egg. For present purposes, the development of the chick has been divided into 3 phases according to the changing requirements at successive stages from fertilisation to hatch.
Phase I. Fertilisation to blastoderm formation. This phase takes place in the oviduct and terminates at oviposition. Gamete interaction occurs within 15 min after ovulation, and the first cleavage division some 4 h later (Perry, 1987). In the following 20 h, subsequent divisions give rise to a simple sheet of cells overlying a subblastodermal cavity (Kochav, Ginsburg and Eyal-Giladi, 1980). During its passage through the oviduct, the ovum is invested with albumen in the magnum, then with the shell membrane in the isthmus where cleavage commences. In the uterus, the albumen is doubled in volume by the absorption of uterine fluid (pumping fluid), and finally the shell undergoes slow calcification. For hens laying in long sequences of one egg per day, oviposition is followed within 15-30 min by the next ovulation and the cycle is repeated.
Phase II. Embryonic morphogenesis. This phase takes place in the first 3 days of incubation of the egg (stages 1-18, Hamburger and Hamilton [1951]). At stage 20, the embryo is 10 mm in length, and the extra embryonic blastoderm extends around the yolk to its equator.
Phase III. Embryonic growth. This phase takes place in the final 18 days of incubation of the egg (stages 18-45, Hamburger and Hamilton [1951]).
Several methods are available for the short-term culture of embryos in Phase II (New, 1966) and for the long-term culture of more advanced embryos (Dunn, Fitzharris and Barnett, 1981; Ono and Wakasugi, 1984; Rowlett and Simkiss, 1985, 1987). Some involve transplantation of the embryo from the yolk, whereas others involve transfer of the embryo and intact yolk to a culture vessel. The latter method provides the more favourable conditions for long-term culture and is used exclusively for the present culture system.